Chronic wounds represent a major health problem for which treatment options remain limited. Chronic wounds are thought to result from a persistence of the inflammatory stage of wound healing and their successful management will require a better understanding of the molecular forces which contribute to poor wound healing. Comparative proteome analysis of normal healing and non-healing wounds has indicated that fibronectin as well as proteases such as MMPs are major components of the non-healing wound exudate. The level of cytokines within the wound bed can affect the balance between proteases and their inhibitors, thereby leading to excessive proteolysis and the continual generation of matrix fragments. Extracellular matrix-derived Damage Associated Molecular Patterns (DAMPs) are products of tissue injury and promote inflammation through the activation of Toll-like Receptors (TLRs). TLR4, originally identified on immune cells as a receptor for bacterial lipopolysaccharide (LPS), is also activated by matrix DAMPs to induce expression of inflammatory cytokines. We have identified two fibronectin Type III domains (FnEDA and FnIII-1c) which activate TLR4 in human dermal fibroblasts and stimulate the synthesis of several pro-inflammatory cytokines, including: CXCL1, 2, and 3, TNF? and IL-8. These two fibronectin Type III domains work synergistically rather than additively to stimulate IL-8 release from dermal fibroblasts, suggesting the existence of ligand specific TLR4 receptor complexes and/or effectors in the molecular pathway initiated by each domain. We hypothesize that TLR4 dependent cytokine induction initiated by the FnEDA and FnIII-1c domains of fibronectin requires domain-specific cell surface TLR4 co-receptors and downstream effectors. This hypothesis will be evaluated in the following two Aims. AIM 1: A non-biased siRNA reverse transfection screen will be used to identify cell surface proteins from dermal fibroblasts and human monocytes which participate in TLR4 mediated cytokine release. AIM 2: Phospho-antibody microarrays will be used to identify proteins which are phosphorylated in response to each Fn(III) domain. These experiments will identify novel co-receptors and signaling intermediates which regulate the inflammatory response to fibronectin DAMPs in both immune and non- immune cells. Dermal fibroblasts do not respond to LPS challenge, suggesting that it may be possible to target DAMP initiated inflammation without affecting the host response to pathogens. The ability to target only DAMP-dependent TLR4 activation represents an innovative approach to the control of chronic inflammation in the wound microenvironment.